Glutathionylspermidine accumulates in Escherichia coli at the end of growth, and accounts for all of the intracellular spermidine and one-half of the intracellular glutathione. Added (S35) glutathione or H3) spermidine are incorporated into glutathionylspermidine. Glutathionylspermidine has been isolated from E. coli and characterized by chemical and enzymatic techniques. Turnover studies have shown that, even when it is accumulating, glutathionylspermidine is in equilibrium with unbound glutathione and with free spermidine. Enzymes are present in E. coli for both the synthesis and the degradation of glutathionylspermidine. Closely related to these studies are our studies showing that glutathione is normally present in large amounts in E. coli. For this purpose, a new analytical technique was developed for the automated amino acid analyzer, based on elution from an anion exchange resin. This technique permits the quantitative determination of free and total glutathione, and of related compounds, in liver and other tissues, as well as in E. coli. Studies on the biosynthesis of spermidine are being continued, particularly the first step, namely, the synthesis of adenosylmethionine.